Microscope and method for microscopic imaging of an object

ABSTRACT

A microscope for imaging an object, comprising a lens assembly, which defines an optical axis and a focal plane perpendicular thereto, and correction optics, which are adjustable for adjustment to a depth position and which correct a spherical aberration on the lens assembly which occurs during imaging of the object at a specific depth position of the focal plane. The microscope may be used to determine a phase difference of radiation from a first lateral region and a second lateral region of the object, and to use a previously known connection between the phase difference and a modification of the spherical aberration caused thereby in order to determine an adjustment value of the correction optics, such that the spherical aberration is reduced when imaging the second region.

PRIORITY CLAIM

The present application is a National Phase entry of PCT Application No.PCT/EP2018/056434, filed Mar. 14, 2018, which claims priority fromGerman Patent Application 10 2017 105 928.8, filed Mar. 20, 2017, thedisclosures of which are hereby incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The invention relates to a method for microscopic imaging of an objectby means of a microscope comprising an objective including a correctionoptical unit. The objective defines an optical axis and a focal planeperpendicular thereto. The correction optical unit of this objective isadjustable to a depth position to correct a spherical aberrationoccurring when imaging the object at this depth position of the focalplane. Further, the invention relates to such microscope.

BACKGROUND OF THE INVENTION

Microscopes often need to have a high degree of flexibility in researchsince very different types of experiments are carried out. Here, theremay be great variation both in the objects to be observed and in theobject carriers. In the case of objectives with a high numericalaperture, deviations of the refractive index along an optical path fromthe focal plane in the object to the objective have a strong influenceon the diffraction-limited imaging capability. One option for adaptingthe spherical aberration associated with imaging the object to differentobject carrier thicknesses, such as, e.g., coverslip thicknesses from0.15 mm to 1.5 mm, lies in setting a correction ring at the objective tothe appropriate value and consequently reducing the spherical aberrationduring the imaging.

Such imaging aberrations become ever more noticeable, the deeper a focalplane lies in the object. Visible spherical aberrations already occur atdepths of a few micrometers if there is a refractive index difference inthe object and the numerical aperture of the objective iscorrespondingly high (e.g., a numerical aperture of 1.2, as isconventional in confocal microscopy). Imaging in three dimensions, i.e.,imaging in thicker or deeper objects, is becoming ever more important,whether in the case of examining cell cultures in 3D, in the case ofspheroids or in the case of thicker slices. Excellent imaging quality isbecoming ever more important for this, and so losses in the quality onaccount of spherical aberration are no longer accepted.

The applications, particularly in the case of fluorescence microscopy,and hence the requirement on the microscope vary greatly. By way ofexample, in one experiment, it may be necessary only to observe thefirst 10 μm from a coverslip surface, while, in another experiment,however, the intention is to image into the object to a depth of 200 μm.Similar considerations apply to the temperature. One experiment iscarried out at room temperature, while another experiment is performedat 37° C. This has an influence on the optical behavior of themicroscope, and hence on the imaging properties thereof.

Until now, it has been cumbersome to exactly set the correction opticalunit, which usually is a so-called correction ring at the objectivematching the requirements of the object carrier or of the object. Theexperimenter must try different settings in order to find the optimalvalue. Moreover, changing the setting during the experiment is virtuallyprecluded.

Prior art discloses various methods or designs for microscopes, whichoperate either in an automated or in a partly automated fashion andcontain an iterative procedure for correcting spherical aberration. US2008/310016 A describes correction of spherical aberration in respect ofa thickness of a coverslip. US 2005/024718 A and JP 2005/043624 A2require the user to enter optical parameters, from which a correction ofspherical aberration is subsequently derived. US 2011/141260 A and US2014/233094 A describe iterative correction methods for sphericalaberration which evaluate the contrast or the brightness in an imageanalysis.

Phase aspects in microscopy are discussed in Humphry: “Opticaltransmission mode imaging with the Phase Focus Virtual Lens”, PhaseFocus Limited, No. TB02, Mar. 22, 2010; in the phase contrast camera“SID4bio” by Phasics S.A.; in Marquet et al.: “Review of quantitativephase-digital holographic microscopy: promising novel imaging techniqueto resolve neuronal network activity and identify cellular biomarkers ofpsychiatric disorders”, Neurophotonics, Vol. 1(2), October-December 2014and in Rappaz et al.: “Simultaneous cell morphometry and refractiveindex measurement with dual-wavelength digital holographic microscopyand dye-enhanced dispersion of perfusion medium”, Optics Letters, Vol.33, No. 7, Apr. 1, 2008.

For the purposes of improving the imaging quality, DE 102014002584 A1ascertains a geometric path length position of an object point with muchoutlay by way of optical coherence tomography. WO 2013/130077 uses SLMsfor illumination and imaging.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method and a microscopefor imaging an object, by means of which the object can be imaged asaberration-free as possible with little outlay.

In a method for imaging an object, use is made of a microscope whichcomprises an objective and an adjustable correction optical unit. Theobjective defines an optical axis and a focal plane perpendicularthereto. The correction unit corrects a spherical aberration at theobjective, said spherical aberration occurring when imaging the object.A set value of the correction unit applies to one certain depth positionof the focal plane. The method comprises the following steps:illuminating the object; capturing radiation reflected or transmitted bythe object; performing quantitative phase contrast imaging fordetermining a phase difference of radiation between a first lateralregion and a second lateral region of the object. Either sphericalaberration of imaging of the object or the spherical aberrationcorrection necessary is known for the first region. A relationshipbetween phase difference and a change in spherical aberration caused bythis phase difference is used to ascertain a set value of the correctionoptical unit such that the spherical aberration is reduced in the secondregion, too. The correction optical unit is set to the set value and theobject in the second region is imaged, then.

Absolute values of the spherical aberration need not be known in themethod. The method relates to a first region, for which the sphericalaberration is corrected or correctable by the correction optical unit.By way of reference to this first region and by ascertaining the phasedifference and by using the relationship between the phase differenceand the change in the spherical aberration caused thereby, thecorrection optical unit can very easily be set such that the object isimaged with as little spherical aberration as possible in the secondregion, too. In embodiments, the correction optical unit is first set insuch a way that the first region is imaged with as little sphericalaberration as possible. By way of example, a region of the object thatis known in terms of its refractive index or an easily accessible regionof a sample can be used to this end. Consequently, the first region canbe selected in such a way that an image with little spherical aberrationor with completely compensated spherical aberration is obtained withlittle outlay and quickly. Then, without further outlay, the methodsupplies a low-aberration or aberration-free image of the second region,too, by virtue of the phase contrast imaging being performed first andthe appropriate setting for the correction optical unit then beingobtained for the second region, taking into account the relationshipbetween the change in the spherical aberration and phase contrast.Consequently, the combination of phase contrast imaging and use of arelationship reproducing the change in the spherical aberration allows aparticularly simple procedure. In a particularly preferred embodiment,the relationship specifies a setting of the correction optical unit as afunction of the phase contrast, more particularly the change in thesetting. Thus, the change in the spherical aberration is directlyconverted into a setting of the correction optical unit.

The phase difference is determined by using quantitative phase contrastimaging. A device for quantitative phase contrast imaging optionallycomprises a light source and a detector for determining the phasedifference. Determining the phase difference in this way is equallypossible in reflected light and in transmitted light microscopy methods.

A microscope for imaging an object comprises an objective, an adjustablecorrection optical unit, a drive, a device for quantitative phasecontrast imaging and a control device. The objective defines an opticalaxis and a focal plane perpendicular thereto. The correction opticalunit corrects a spherical aberration at the objective, said sphericalaberration occurring during the imaging of the object. A set value ofthe correction optical unit applies to one certain depth position of thefocal plane. The drive sets the correction optical unit. The device forquantitative phase contrast imaging is embodied to illuminate the objectwith radiation, to capture the radiation reflected or transmitted by theobject and to determine a phase difference of radiation between a firstlateral region and a second lateral region. Pre-stored in the controldevice is a relationship between the phase difference and a change inthe spherical aberration caused thereby. On the basis of thisrelationship, the control device controls the drive in such a way thatthe spherical aberration in the focal plane is reduced also in thesecond region.

As a result of the present invention, spherical aberration present whenimaging the object is automatically corrected for a “fresh” secondregion based on a “known” first region. In embodiments, this is done byevaluating a phase difference between two lateral regions in the objectbefore or during the imaging of the object and by obtaining of acorrection of the spherical aberration for the second region.Accordingly, the experimenter need not consider whether the correctionoptical unit is acceptably set to correct spherical aberration for thesecond region of the respective object. Moreover, it is now possible tobe able to suitably set the correction optical unit, even during anexperiment, for objects that have different refractive indices alongtheir lateral extent and consequently cause different sphericalaberrations. By way of example, the correction optical unit can be setaccording to the lateral region in which scanning is currently carriedout during scanning imaging of the object, and so the sphericalaberration is minimized for the overall imaging.

A further advantage is that determining the phase difference byquantitative phase contrast imaging causes little or no damage to theobject since the radiation used to this end does not interact with theobject, or only interacts weakly therewith.

By way of example, the method can be carried out with the aid of thecontrol device of the microscope. By way of example, the control devicecan be embodied as a microprocessor, an electric circuit, a computer orany other programmable apparatus.

The microscope used for the method can carry out different imagingmethods, depending on embodiment. By way of example, the microscope maybe embodied for wide-field imaging and/or for scanning imagingtechniques, such as confocal microscopy. Further, it is optionallypossible to use the microscope to capture fluorescence images of theobject.

The object optionally comprises the sample that, in fact, should beimaged and a mounting medium, that surrounds the sample, or theembedding medium. By way of example, the object comprises a cell cultureto be imaged as a sample and the solution in which the cell culture isembedded as a medium.

The objective serves to image the object, but can be also used forillumination purposes at the same time. The objective has an opticalaxis. The focal length sets the depth position of the focal plane.Optionally, the objective and/or an object carrier are provided with az-drive, and set the position of the focal plane in the object. Thez-drive may be connected to the control device such that the controldevice is able to set the depth position of the focal plane. Moreover,the control device optionally captures the current position of the focalplane, for example with the aid of the position adopted by the z-drive.

The correction optical unit serves to correct the spherical aberrationwhich occurs during the imaging of the object. The correction opticalunit may be the above-described correction ring at the objective;however, it is also possible that optical elements at a distance fromthe objective are used, said optical elements facilitating themodification of the spherical aberration of the imaging by means of theobjective. By way of example, the correction optical unit can bearranged on the detector side. The correction optical unit may compriseoptical elements which deflect radiation differently depending on theirposition in relation to the optical axis.

The correction optical unit is set with the aid of a drive which isconnected to the control device, either by lines or in a wirelessmanner. The control device may capture the position of the drive andthus the current set value of the correction optical unit.

The phase difference caused by the radiation passing through the objectfrom the first region and the second region is proportional to anoptical path length difference which the radiation from the first regionand from the second region travels to the detector in each case. Thisoptical path length difference is responsible for the sphericalaberration. The phase difference satisfies following equation (1):

${\Delta\varphi} = {\frac{2\pi}{\lambda}\left( {n_{2} - n_{1}} \right)d}$

Here, Δφ is the phase difference, λ is the wavelength of the radiationfor determining the phase difference, n2 is the refractive index in thesecond region, n1 is the refractive index in the first region and d is apath length. A path length difference, which emerges from equation (1)but without the prefactor 2π/λ, is analogous to the phase difference Δφ.The path length d is the region, e.g., the thickness of the sample, fromwhich radiation contributes to the imaging. The thickness, or therefractive index of the sample can be determined from the phasedifference measurement using the equation specified above. The samplethickness can be derived from the phase data if the refractive index isconstant and known. The refractive index can be derived if the thicknessis known (and constant). The depth of field range of the objective isrelevant for this purpose in some embodiments, specifically when, interalia, the sample is very much thicker than the depth of field range. Inother embodiments, in which the sample is thinner than the depth offield range and lies completely in the depth of field range, the samplethickness is decisive for the path length d. If wavelength and pathlength d are constant, the phase difference only depends on thedifference in the refractive indices between the first region and thesecond region.

The relationship links the difference between the refractive index and aspherical aberration caused thereby with the phase difference or theoptical path length difference. The relationship can be a formula or atable. The relationship is optionally determined once by determiningvery different refractive indices for the object and stored as a table;this calibration can also be regularly repeated for the microscope. Thevery different values of the phase difference can be ascertained to verygood approximation by way of interpolation.

In embodiments, the path length d is included as a constant factor inthe relationship. Therefore, a specification about this (constant) pathlength d is used in embodiments for the purposes of performing themicroscopy method or in the microscope. This specification can beobtained differently for various embodiments. In some embodiments, thepath length d equals the sample thickness if the depth of field range ofthe employed objective is greater than the sample thickness and thesample thickness lies completely in the depth of field range. In otherembodiments, in which the conditions are virtually inverted and thesample is very much thicker than the depth of field range, this does notapply. However, the sample thickness varies over the object field insuch embodiments. By way of example, the thickness d can be defined inthe case of histological sections or by the dimensions of a microfluidicchannel, or else be determined by other methods. Here, it is possible toascertain a “mean” refractive index of the sample, for example with alow-magnification objective, and then subsequently use this parameterfor correcting the spherical aberration in the case of a fluorescencemeasurement with a high-resolution objective. Additionally, the depth offield range can be approximately assumed as value for d.

In embodiments, it is also possible to perform a phase difference orpath length difference measurement in the microscope at an interfacebetween a biological sample to be examined by microscopy and a medium,for example a culture medium, surrounding the latter. Phase differencemeasurements or path length difference measurements are known inmicroscopy (see the publications specified below) and it poses noproblem to a person skilled in the art to configure a microscope forsuch measurement. Here, the phase difference between the sample and thesurrounding medium is ascertained for the purposes of determining thepath length d (ascertaining the path length difference is equivalentthereto). Two measurements are carried out, in which the phasedifference (or path length difference) differs in that the refractiveindex of the surrounding medium is different. In a first variant, thisis implemented by virtue of the surrounding medium being dispersive,i.e., exhibiting a wavelength dependence on the refractive index, andthe two measurements being carried out at different wavelengths. In asecond variant, the medium is replaced by a medium with a differentrefractive index between the two measurements. The path lengthdifference (or phase difference) emerges from the product of path lengthd and refractive index difference between medium and sample in each ofthe two measurements in both variants. Consequently, two equations withtwo unknowns are obtained; this system of equations is solvable withoutproblems and it is possible to ascertain both the path length differenceand the refractive index (unknown a priori) of the sample. Knowledge ofthe path length d now renders it possible, without interpolation, to useor determine, to a very good approximation, the various values of thephase difference, even outside of the interface between sample andmedium used for ascertaining d.

On account of the relationship, the control device ascertains a setvalue for appropriately setting the correction optical unit. The setvalue optionally depends on the current position of the correctionoptical unit and the desired position ascertained from the relationship.By way of example, the current set value of the correction optical unitis known from knowledge of the spherical aberration in the first region.Further, the set value may also specify an absolute position of thecorrection optical unit. The object is imaged in the second region aftersetting the correction optical unit.

In one development, it is preferable for the relationship to be modifiedin respect of a penetration depth. The penetration depth is the depth atwhich the focal plane lies under the sample surface, as seen in theimaging direction. It is particularly relevant when a high penetrationdepth is used and the depth of field range of the objective is low (highnumerical aperture), since the spherical aberration increases withpenetration depth. Therefore, measuring the penetration depth ispreferred, wherein measuring the penetration depth optionally comprisesdetecting an interface between an object carrier and the object andcapturing a position of the focal plane. In embodiments, the penetrationdepth is relevant for the path of the radiation from the interface tothe objective. Various options are conceivable for determining thepenetration depth. By way of example, if the distance between the objectcarrier and the objective and the focal length of the objective areknown, the penetration depth can be ascertained from this difference.Moreover, it is possible to determine the penetration depth on the basisof the object. Particularly preferably, however, the interface betweenthe object and the object carrier is determined, for example by virtueof capturing the reflection of the radiation caused at the interface ifthe focal plane of the objective coincides with the interface. Since thecontrol device can preferably capture the focal length of the objectiveby way of the z-drive, the position of the interface is detectable.Then, the penetration depth is captured by virtue of the adjustment ofthe focal length of the objective being recorded by means of the z-driveand the current focal plane consequently being known. Then, thepenetration depth is the difference between the current focal plane andthe interface. It is further possible to obtain a tomographic phaseimage, for example by virtue of the phase differences being measured indifferent z-planes (focal planes). Consequently, an xyz-phaserelationship of the sample is obtained. From this, a z-averagedrefractive index can now be determined for each penetration depth. Byway of example, if the intention is to carry out a fluorescencemeasurement at a penetration depth of 20 μm, the refractive indices overthe first 20 μm can be averaged and included in the correction of thespherical aberration. If one is situated 100 μm deep within the sample,it is necessary to average over 100 μm.

If the object is imaged at different depth positions, it is preferablefor a plurality of phase differences to be determined in a plurality ofobject planes that are spaced apart from one another. By way of example,a thick sample is captured in a plurality of z-positions by way of aso-called z-stack. Recording the phase differences in a plurality ofobject planes, i.e., by way of a z-stack, is particularly helpful if theprecise positions of the focal planes for imaging the object are not yetknown when determining the set values. However, it is also possible forthe phase difference to be measured first for each focal plane to beimaged in the object, for the spherical aberrations then to be correctedand for this focal plane to be subsequently imaged. Subsequently, thefocal plane is displaced, the phase difference is measured anew, thespherical aberration is corrected where necessary and, following this,the object is imaged in the modified focal plane.

In embodiments, a holographic quantitative phase contrast imaging methodknown as digital holographic microscopy is used for determining thephase difference. Coherent or partly coherent radiation can be used forthe phase contrast imaging. Further, it is possible to use at least twowavelengths for the quantitative phase contrast imaging, the object,more particularly the medium, having a significant refractive indexdifference, which is known in advance, at said wavelengths. Thequantitative phase contrast imaging optionally is a ptychographicmethod. A wavefront sensor is used for the quantitative phase contrastimaging in a development of the microscope. Additionally, aninterferometric structure can be used for the quantitative phasecontrast imaging. The light source for the quantitative phase contrastimaging may comprise a spatial light modulator or the phase of theradiation used for the quantitative phase contrast imaging is optionallyvaried. Further, the radiation for the quantitative phase contrastimaging can be produced from various illumination angles. Examples for adevice for quantitative phase contrast imaging or methods carried outtherewith are described in U.S. Pat. No. 7,948,632 B2, Wang et al.:“Spatial light interference tomography (SLIT)”, Optics Express, Vol. 19,No. 21, Oct. 10, 2011, and Tian et al.: “Quantitative differential phasecontrast imaging in an LED array microscope”, Optics Express, Vol. 23,No. 9, May 4, 2015.

As already mentioned, the phase difference caused by the object ismeasured between two lateral regions that are spaced apart from oneanother in an object plane. The object plane is identical to the focalplane or parallel thereto. Correcting the spherical aberration becomesever more precise the closer the object plane lies to the focal planefor imaging the object. By way of example, determining the phasedifference and subsequent imaging of the object are carried out by thesame objective, with the object plane lying in the focal plane. However,it is also possible for the phase difference to be determined once in acertain object plane and for the object to be subsequently imaged indifferent focal planes. Nevertheless, determining the phase differenceonce in an object plane represents a very good approximation for thephase difference in the focal plane. The lateral regions between whichthe phase difference is determined can be approximately punctiform,particularly if the microscope carries out a scanning imaging method. Ifan imaging method is used for quantitative phase contrast imaging, it ispossible to simultaneously capture not only two lateral regions but aplurality of lateral regions.

In embodiments, the first region is chosen in such a way that thespherical aberration thereof is known or compensated. By way of example,this comprises the spherical aberration for imaging the object from thefocal plane in the first region already having been minimized. By way ofexample, this is the case if the correction optical unit is set for aregion of the object with a certain refractive index as a standardvalue; this can, inter alia, be implemented automatically. By way ofexample, such a standard value may be suitable for water or an aqueoussolution, which surrounds samples to be imaged, such as a cell or anyother biological samples. Knowledge of the setting for compensating thespherical aberration in the first region optionally comprises knowledgeabout the set value for the correction optical unit, for which thespherical aberration is minimized, for example. In order to ascertainthe first region, the object is optionally initially imaged without afurther correction of the spherical aberration in order to identifywhere regions with known refractive index are present. Further, thespherical aberration may be corrected manually—also with the aid of thecontrol device and the drive—until the imaging of the object is optimalin the first region. Moreover, the first region may also be such aregion in which the sample to be imaged is not present in the object andthe first region is then measured for air. Consequently, the objectoptionally comprises the sample to be imaged and also the surroundingsthereof.

In particular, the second region is the region of the object that is ofinterest for the examination to be carried out. As a rule, the secondregion has a refractive index that differs from the refractive index inthe first region because otherwise there would be no need for correctingthe spherical aberration. Usually, the refractive index in the secondregion is unknown, and so purely determining the phase differencebetween the first region and the second region helps to optimize thespherical aberration for imaging the entire object.

In order to simplify and/or accelerate the correction of sphericalaberration, one development prefers for a third lateral region of theobject to be used, for which the same refractive index, within atolerance range, is present as for the second region, wherein the setvalue for the second region is also used for imaging the third region.By way of example, the third lateral region can be a region in the focalplane of the object, for which it is expected that the refractive indexis virtually identical to the refractive index in the second region. Byway of example, the second region and the third region may be within acell such that the refractive index for both regions, and hence also thespherical aberration for the imaging thereof, are approximately thesame. By way of example, the refractive indices of the various relevantsample regions can be averaged in order to correct a sphericalaberration for the sample region. Consequently, an acceptable correctionof the spherical aberration can be provided with a reduced number ofdetermining phase differences. Further, this also allows increase in themeasurement speed for imaging the object since a time-consumingadjustment of the correction optical unit is dispensed with during theimaging of the object if the same set value is used for the third regionas for the second region. A similar statement applies to z-stackrecordings. If a sufficient number of similar phase difference values orrefractive indices are obtained in the various z-planes (in comparisonwith the z-plane lying therebelow), the spherical aberration need not becorrected in each z-plane. A similar statement applies to the z-stack byz-stack ascertainment of the phase difference of an object. This meansthat if similar phase difference values or refractive indices areobtained in the various z-planes, the spherical aberration need not becorrected in each z-plane. Optionally, an additional correction is onlycarried out once a maximum deviation has been exceeded.

In order to design the relationship even more precisely and consequentlyfacilitate an even more accurate correction of the spherical aberration,it is preferred in a development for at least one parameter to becaptured, said parameter comprising a temperature of the object, amaterial of an object carrier, an object carrier thickness, an immersionmedium for the objective or a wavelength of radiation for imaging theobject, with the relationship being modified in respect of the at leastone parameter. It is known that the refractive index of a materialdepends on its temperature. Consequently, the spherical aberration whenimaging by means of the objective is also dependent on temperature. Bycapturing the temperature of the object, for example by means of asensor or by way of an entry, for example if the temperature of theexperiment is fixedly predetermined, it is possible for the relationshipto be modified such that a different set value arises in the case ofdifferent temperatures. Consequently, the correction optical unit is setin a temperature-dependent manner. Here too, the relationship may bedetermined by calibration in respect of the temperature dependence. In adevelopment, the temperature of the object is determined by capturing anenergy parameter of radiation for illuminating the object and byascertaining the temperature of the object from the energy parameter. Byway of example, the energy parameter may be the intensity or the powerof the illumination radiation. This may be ascertained in the objectitself or by output coupling a measurement component of the illuminationradiation from the illumination beam path. To this end, use can be madeof a power detector or an intensity detector. The temperature in theobject may be calculated on the basis of the energy parameter and theexpected or known absorption of the illumination radiation by theobject. The type of object may also be used for calculating thetemperature, namely in respect of the strength of the absorptionbehavior of the respective object. In this way, the relationship can beautomatically modified in respect of the temperature.

Along the path traveled by the radiation from focal plane to theobjective, the radiation passes both an object carrier or coverslip andalso, under certain circumstances, an immersion medium. These structureshave a refractive index that contributes to the spherical aberration ofthe imaging of the object. By capturing an object carrier thickness orcoverslip thickness and/or the refractive index of the object carrierand/or of the immersion medium, in each case by way of example byentering an associated specification, the relationship can be improvedand modified in respect of the spherical aberration, which is changed bythese aspects.

Since the spherical aberration depends on the refractive index, dependson the wavelength in the case of dispersive materials, the sphericalaberration then is likewise wavelength-dependent. The wavelength of theradiation for imaging the object is consequently a further parameter inrespect of which the relationship may be modified in order to improvethe setting of the spherical aberration. The wavelength may be enteredmanually; the wavelength of the induced fluorescence radiation is known,particularly in the case of fluorescence imaging, to the controllerand/or the user. Moreover, the microscope may be provided with awavelength sensor, by means of which the wavelength of the imagingradiation can be determined in an automated manner. Optionally, theillumination wavelength and/or filter settings can be taken intoaccount.

In a development, the method can be used to determine the refractiveindex of the object in the second region. This was already explainedabove with reference to equation (1). Consequently, this developmentrenders it possible to determine the refractive index in the sample; byway of example, should the refractive index of an aqueous mediumsurrounding a cell culture be known, it is consequently possible todetermine the refractive index in the cell culture itself. It can thenbe used to set the correction optical unit.

In order to protect the sample from radiation damage, it is preferablefor the phase difference to be determined by means of quantitative phasecontrast imaging, with the radiation for the quantitative phase contrastimaging optionally lying in the infrared range. Quantitative phasecontrast imaging is known for causing little radiation damage in theobject. This is particularly successful if infrared radiation is used.Further, the use of infrared radiation is advantageous in that aninteraction with some fluoresced substances can be avoided within thescope of fluorescence imaging.

It goes without saying that the aforementioned features and those yet tobe explained below can be used not only in the combinations specifiedbut also in other combinations or on their own, without departing fromthe scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is explained in even greater detail below for example withreference to the accompanying drawings, which also disclose featuresessential to the invention. In the figures:

FIG. 1 shows a schematic view of the microscope;

FIG. 2 shows an objective and an object carrier, with an object arrangedthereon, of the microscope in FIG. 1;

FIG. 3 shows a magnified illustration of an embodiment of the objectcarrier with the object;

FIG. 4 shows a schematic illustration of the object; and

FIG. 5 shows a block diagram illustrating a method for imaging theobject.

DETAILED DESCRIPTION

A microscope 10 facilitates imaging of an object 12. The radiationreflected or transmitted by the object 12 is collected by an objective14 and imaged on an imaging detector 16 by means of an imaging beam path18. The objective 14, the imaging detector 16 and/or the imaging beampath 18 are arranged within the housing 20 of the microscope 10. Theimaging beam path 18 may be embodied for various types of imaging of theobject 12. By way of example, the object 12 can be recorded in the widefield or by means of a scanning imaging method. Further, the microscope10 may be embodied for fluorescence measurements. Depending on the typeof employed imaging, the imaging beam path 18 comprises various opticalelements and/or further components, such as, e.g., a scanning device fordeflecting radiation. These components are not illustrated in FIG. 1.The object 12 can be illuminated, for example with a reflected-lightlight source or with the transmitted-light light source 22 illustratedin FIG. 1. By way of example, the light source 22 may produce whitelight or emit radiation in a certain wavelength range suitable forfluorescence microscopy.

The imaging detector 16 is embodied to convert an optical image of theobject 12 produced thereon by means of the imaging beam path 18 intoelectrical signals, which are transmitted to a control device 24 via aline. From the electrical signals provided by the imaging detector 16,the control device 24 produces an electronic image which, for example,is displayed to the experimenter on a display device 26, such as amonitor, connected to the control device 24.

The object 12 is arranged on an object carrier 28 which has a certainobject carrier thickness OD. By way of example, the object carrier 28may be a glass plate. Further, the object carrier 28 may be the base ofa Petri dish made of glass or plastic. The object carrier 28 can bemounted in movable fashion in relation to the housing 20 such that theobject 12 is displaceably mounted in relation to the objective 14.

Optionally, the microscope 10 comprises a plurality of objectives 14,which are arranged on a revolver 30. The revolver 30 can be used topivot-in the objective 14 which is intended to be used for the imaging.The revolver 30 can be adjusted manually or be moved by a revolver driveconnected to the control device 24. Neither the revolver drive nor theconnection to the control device 24 are illustrated in FIG. 1. At leastone of the objectives 14 is provided with a correction optical unit 32.The correction optical unit 32 is adjustable and serves to modify aspherical aberration of the imaging of the object 12. Depending on thedepth position of a focal plane in the object, there is a change in thespherical aberration, and so the spherical aberration when imaging theobject 12 may be minimized by an adjustment of the correction opticalunit 32. The objective 14 illustrated on the right in FIG. 1 and theobjective 14 illustrated in FIG. 2 comprise a correction ring as acorrection optical unit 32. It is securely connected to the objective 14and rotatable by way of a drive 34. The drive 34 is connected to thecontrol device 24 by means of a line or via radio, with the connectionnot being illustrated in FIGS. 1 and 2. Consequently, the control device24 is able to actuate the drive 34 and able to influence the sphericalaberration of the imaging of the object 12. Optionally, the controldevice 24 may capture the current setting of the correction optical unit32 by means of the drive 34.

Alternatively, the correction optical unit 32 can be arranged in amanner detached from the objective 14. In the objective 14 illustratedto the left in FIG. 1, the correction optical unit 32 is arranged in theimaging beam path 18. It, too, is provided with a drive 34.

Further, the microscope 10 comprises a device for quantitative phasecontrast imaging 36. The device for quantitative phase contrast imaging36 may comprise very different types, by means of which quantitativephase contrast imaging can be performed. By way of example, reference ismade here to the methods described in the literature referencesmentioned further above.

The device for quantitative phase contrast imaging 36 is embodied todetermine a phase difference between a first lateral region 40 and asecond lateral region 42; as a rule, these lateral regions lie within acommon object field 54 of the objective 14. This is illustrated, interalia, in FIG. 2. The device for quantitative phase contrast imaging 36comprises an optional illumination device and a phase detector or phasecamera 44. By way of example, the illumination device produces whitelight or infrared light, which is imaged on the phase detector 44 bymeans of the objective 14 and the correction optical unit 32. In orderto output couple radiation from the imaging beam path 18 to the phasedetector 44 for the purposes of detecting the phase difference,provision is made of a beam splitter 46. The phase detector 44 detectsthe phase difference between radiation from the first region 40 and thesecond region 42. Preferably, the device for quantitative phase contrastimaging 36 is embodied to determine phase differences between the firstregion 40 and a plurality of second regions.

The control device 24 is connected to a storage device 48, in which arelationship between the phase difference and a change in the sphericalaberration caused thereby is saved. This relationship may have beendetermined by a preceding calibration and represents, for example, atable. The storage device 48 can be embodied as part of the controldevice 24, which may comprise a microprocessor, computer or the like;the storage device 48 may be a writeable memory, such as a RAM (randomaccess memory). The control device 24 calls the relationship from thestorage device 48 and thus determines a set value for the correctionoptical unit 32, by means of which the spherical aberration for imagingthe object 12 in the second region 42 is reduced. By way of example, therelationship may contain the change in the spherical aberration in theform of a setting of the correction optical unit, for example thecorrection ring. Here, this may be specifying a change. In particular,the relationship may have been ascertained by earlier experiments usinga sample with a known refractive index distribution. Alternatively, itcan be obtained by calculation from the structure of the correctionoptical unit. Then, the control device 24 actuates the drive 34according to the set value such that the spherical aberration of theimaging of the object 12 in the second region 42 is reduced, preferablyminimized or even completely compensated. Then, the object 12 is imagedin the second region 42.

Further, the microscope 10 comprises an interface 50, by means of whicha plurality of parameters of the experiment can be entered and canconsequently be made available to the control device 24. By way ofexample, the interface 50 is a keyboard or mouse, and it is connected tothe control device 24.

FIG. 4 schematically shows the object 12, which consists of a biologicalsample 52 in an embedding medium 53 in this case. Further, the regions40 and 42, in which the phase contrast measurement is implemented, areplotted schematically. F1 and F2 denote two different focal planes, onwhich the objective 14 can be set. The thickness of the dashed focalplanes elucidates the depth of field range. If a homogeneous refractiveindex of the sample 52 is assumed, the goal is to carry out a differentcorrection for the lateral region 40 than for the lateral region 41 ifthe focal plane F2 is present since the lateral region 40 there iscompletely filled by the sample and its refractive index; by contrast,the lateral region 42 is not. This situation may always occur when asample 52 should be examined by microscopy, the height H of said samplebeing very much greater than the depth of field range and said samplemoreover not having a constant thickness over its lateral extent.Therefore, provision is made in embodiments for the phase differences indifferent planes between objective and desired object plane, for examplecorresponding to F1, to be ascertained and for the interposed layersbetween the coverslip 28 and the actual focal plane F1 desired in theobject, captured thereby, to be taken into account for the correction.If only the focal plane F1 is considered, it can be seen that no phasedifferences arise in the lateral regions 40 and 42 as a result of thegeometry of the sample 52 (assuming a homogeneous refractive index ofthe sample 52). By contrast, a phase difference is obtained in the focalplane F2. Therefore, provision is made in embodiments for the geometryof the object 12, which, e.g., comprises a spherical sample 52 with adiameter of, e.g., 150 μm, to be taken into account and for a correctiondepending on the lateral position (40, 42) and the position of the focalplanes (F1, F2) to be undertaken with knowledge and followingcalibration of the refractive index of the sample 52.

A method for imaging the object 12 is shown in a block diagram in FIG.5. An object carrier thickness OD is entered, for example with the aidof the interface 50, in an optional step S1. The spherical aberration ofthe imaging of the object 12 in the first region 40 is optimized in astep S2. To this end, an appropriate algorithm is saved in the controldevice 24. This algorithm can use the refractive index in the firstregion 40 of the object 12. By way of example, the first region 40 isarranged in a section of the object 12 in which a known medium, e.g.,water or an aqueous solution, is situated. By way of example, this canbe the medium of a cell culture or any other biological sample. Therefractive index of the medium is known. The correction optical unit 32is automatically set for minimizing the spherical aberration inembodiments with the aid of the optional object carrier thickness OD andthe refractive index of the object 12 in the first region 40.Alternatively, the optimization of the spherical aberration of theimaging of the object 12 in the first region 40 can be implementedmanually, for example by adjusting the correction optical unit 32.However, according to these steps, this minimization only relates toaberration caused by the first region 40, i.e., the medium.

An object plane, in which a phase difference between the first region 40and the second region 42 should be ascertained, is set in a step S3.This also sets a distance between the object plane and the objectcarrier 28. As a rule, the object plane coincides with the focal plane.

The second region 42 of the object is set in a step S4. This relates tothe region that should subsequently be imaged by means of the microscope10, i.e., for example, a sample situated in the medium. By way ofexample, to this end, the object 12 may be imaged with a non-correctedspherical aberration in an optionally preceding intermediate step inorder to identify where the second region 42 of interest of the object12 is situated.

The phase difference between the first region 40 and the second region42 is recorded in a step S5. Optionally, the phase difference can bedetermined in a plurality of object planes that are spaced apart alongthe optical axis.

Additional parameters in respect of the object 12 are captured in anoptional step S6. By way of example, these may be made available bymeans of an interface 50 of the control device 24. By way of example,the temperature of the object 12, the previously optionally enteredobject carrier thickness OD, a thickness of the object 12, a material ofthe object carrier 28 and/or the refractive index in the first region 40or in the second region 42 are entered. By way of example, thetemperature can be captured by way of a sensor not illustrated here orit may be known for the experiment, for example 37° C. By way ofexample, the thickness of the object 12 can be measured prior to theexperiment or it is known, as is conventional, for example, in the caseof objects 12 in the form of sections. The refractive index for thefirst region 40 may likewise be known, for example because this relatesto an aqueous solution. Further, the refractive index of the object 12may also be known in the second region 42. Moreover, a penetrationdepth, which specifies the distance between the focal plane and aninterface of the object carrier 28, can also be measured in step S6. Tothis end, it is possible, for example, to adjust the depth position ofthe focal plane until a reflection becomes visible at the interfacebetween the object carrier 28 and the object 12. As a result, thedistance to the object carrier 28 is captured for a given depth positionof the focal plane, and so the penetration depth is known. Further, theimmersion medium between the objective 14 and the object carrier 28 canbe taken into account as a parameter.

As already explained in the general part of the description above, thepath length and/or the refractive index in the second region 42 isdetermined from the phase difference in an optional step S7.

Alternatively, the thickness of the sample, if it is less than a depthof field range of the objective, or the depth of field range dimension,optionally corrected by a correction factor, can be used instead of d ifthe sample completely covers the depth of field range. d can also beused to determine the refractive index of the object 12 in the secondregion 42. The set value can equally be ascertained on this basis.

In step S8, the set value for the correction optical unit 32 isascertained on the basis of the relationship. The relationship can bemodified by the penetration depth, which was established previously,such that the set value is even more precise. The relationship ismodified in respect of the penetration depth if, in particular, theobject plane in which the phase difference was determined does notcoincide with the focal plane intended to be imaged. To this end, thedetermination of the refractive indices from different object planesbelow the focal plane (in the case of an inverted microscope) can beincluded inter alra, for example by way of averaging the refractiveindices along the optical axis. Then, it is not only the refractiveindex in the focal plane that is taken into account, but also theinformation from further planes.

In a step S9, the correction optical unit 32 is set according to theadjustment value. As a result, the spherical aberration for imaging theobject 12 is minimized in the second region 42. The object 12 is imagedin the second region 42 in a subsequent step S10.

The phase difference ascertained between the first region 40 and thesecond region 42 is also used as the phase difference between a thirdregion and the first region 40 in an optional step S11. This isimplemented, in particular, if the expectation is that the refractiveindex is identical or virtually the same for the second region 42 andthe third region. Consequently, the object 12 can be imaged withcorrected spherical aberration not only in the second region 42 but alsoin the third region in a subsequent imaging step. A similar statementapplies to recordings in different focal planes, in which the refractiveindex difference (along the optical axis) is similar enough. By way ofexample, this may be set by way of a threshold.

The invention claimed is:
 1. A method for microscopic imaging of anobject, the method comprising the following steps: providing amicroscope comprising an objective which defines an optical axis and afocal plane perpendicular thereto, and which comprises a correction unitfor correcting spherical aberration for one aberration corrected depthposition of the focal plane, wherein the correction unit is adjustableto set the aberration corrected depth position of the focal plane;illuminating the object; capturing radiation reflected or transmitted bythe object; performing quantitative phase contrast imaging in a firstlateral region and in a second lateral region of the object anddetermining a phase difference value between radiation from the firstlateral region and from the second lateral region of the object;providing a relationship between phase difference and change inspherical aberration; determining a set value of the correction opticalunit by utilizing the relationship and the phase difference value, andadjusting the correction optical unit to the set value and imaging theobject in the second region.
 2. The method as claimed in claim 1,wherein the performing step comprises using information on one of thefollowing parameters for the first region of the object: sphericalaberration of imaging, refractive index of the object, and setting ofthe correction optical unit required to correct spherical aberration inthe first region.
 3. The method as claimed in claim 1, wherein thedetermining step comprises ascertaining a path length being an objectdepth region from which radiation from the object contributes to theimaging in the first or second region.
 4. The method as claimed in claim3, wherein the path length ascertaining step comprises varying aneffective refractive index of the object in the first region andmeasuring the phase difference for at least two values of the refractiveindex of the object in the first region and determining the path lengthfrom the measured phase difference.
 5. The method as claimed in claim 4,comprising varying the effective refractive index by using a dispersivemedium in the first region and measuring the phase difference at twodifferent wavelengths.
 6. The method as claimed in claim 3, comprisingusing a thickness of the object as the path length.
 7. The method asclaimed in claim 1, wherein determining the phase difference valuecomprises determining a plurality of phase difference values in aplurality of object planes that are spaced apart from one another alongthe optical axis.
 8. The method as claimed in claim 1, wherein further athird lateral region of the object is imaged, wherein the set value forthe second region is also used for imaging the third region.
 9. Themethod as claimed in claim 1, wherein at least one of the followingparameter values is captured: a temperature of the object, a material ofan object carrier, an object carrier thickness, an immersion mediumutilized in imaging, and a wavelength of radiation used for imaging theobject, wherein the step of providing the relationship includesmodifying the relationship depending on the at least one parametervalue.
 10. The method as claimed in claim 1, wherein the quantitativephase contrast imaging step includes quantitative phase contrast imagingin the infrared spectral range.
 11. The method as claimed in claim 1,wherein the set value is determined on a basis of the phase differencevalue in at least one first object plane and the object is imaged usingthe set value in another, second object plane.
 12. A microscope forimaging an object, comprising: an objective which defines an opticalaxis and a focal plane perpendicular thereto, and which comprises acorrection unit for correcting spherical aberration for one aberrationcorrected depth position of the focal plane, wherein the correction unitis adjustable to set the aberration corrected depth position of thefocal plane; a drive which adjusts the correction optical unit regardingthe aberration corrected depth position of the focal plane; aquantitative phase contrast imaging device configured to illuminate theobject, to capture radiation reflected or transmitted by the object andto determine a phase difference value between radiation from a firstlateral region and from a second lateral region; and a control deviceconfigured to control the quantitative phase contrast imaging device andincluding a pre-stored relationship between phase difference and changein spherical aberration, wherein the control device is furtherconfigured: to determine a set value of the correction optical unit fromthe relationship and the phase difference value such that the sphericalaberration in the second region is reduced, to control the drive toadjust the correction optical unit to the set value, and to image theobject in the second region.
 13. The microscope as claimed in claim 12,wherein the quantitative phase contrast imaging device is configured touse information on one of the following parameters in the first regionof the object: spherical aberration of imaging, refractive index of theobject, and setting of the correction optical unit required to correctspherical aberration in the first region.
 14. The microscope as claimedin claim 12, wherein the quantitative phase contrast imaging device isconfigured to determine a plurality of phase difference values in aplurality of object planes that are spaced apart from one another alongthe optical axis.
 15. The microscope as claimed in claim 12, comprisinga determining unit configured to capture at least one of the followingparameter values: a temperature of the object, a material of an objectcarrier, an object carrier thickness, an immersion medium for theobjective, and a wavelength of radiation utilized for imaging theobject, wherein the relationship depends on the at least one of theparameter values.
 16. The microscope as claimed in claim 12, wherein thequantitative phase contrast imaging device is configured forquantitative phase contrast imaging in the infrared spectral range.